FRET Efficiency Calculator

Calculate FRET efficiency, donor-acceptor distance, and Förster radius for FRET experiments.

Calculate

Donor intensity without acceptor I_D (counts)

Donor intensity with acceptor I_DA (counts)

FRET efficiency E (0-1)

Förster radius R₀ (nm) (nm)

FRET pair dependent: 2-10nm

Donor-acceptor distance r (nm) (nm)

0=calculate from E

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Why the Sixth Power Matters So Much

FRET efficiency depends on the inverse sixth power of donor–acceptor separation, which makes it extraordinarily distance-sensitive.

E = R₀⁶ / (R₀⁶ + r⁶) r = R₀(1/E − 1)^1/6

r relative to R₀	Efficiency
0.5 R₀	98.5%
R₀	50% by definition
1.5 R₀	8%
2 R₀	1.5%

Doubling the distance reduces efficiency 64-fold. This is why FRET works as a molecular ruler over roughly 0.5 to 1.5 times R₀ — typically 2 to 10 nm — and gives essentially no information outside that window.

That range happens to match the dimensions of protein-protein interactions and conformational changes, which is why the technique is so widely used despite its narrow window.

What Determines R₀

Factor	Effect
Spectral overlap	Donor emission must overlap acceptor absorption
Donor quantum yield	Higher yield raises R₀
Orientation factor κ²	Ranges 0 to 4; assumed 2/3
Refractive index	Minor

The orientation factor is the main source of uncertainty. It is conventionally assumed to be 2/3, which holds only if both dipoles rotate freely during the excited-state lifetime. Rigidly held fluorophores can give κ² anywhere from 0 to 4, and since R₀ depends on its sixth root, the distance error can be substantial.

Measuring Efficiency

Donor quenching is the simplest approach but requires a matched sample lacking acceptor. Acceptor photobleaching is more robust: bleach the acceptor and the donor signal recovers in proportion to the FRET that was occurring, providing an internal control within the same cell.

Fluorescence lifetime imaging is more rigorous still, since donor lifetime shortens with FRET and is independent of concentration and excitation intensity — the two largest sources of artefact in intensity-based measurements.

Worked Examples

Example 1: Donor quenched: I_D=100, I_DA=35

E=1-35/100=0.65

Result: 65% FRET, distance=R0×(0.35/0.65)^(1/6)=0.91R0

If R0=5nm: distance=4.5nm

Example 2: Distance from E=0.5 (50% FRET)

r=R0×(0.5/0.5)^(1/6)=R0×1

Result: r=R0 exactly at 50% FRET

FRET is most sensitive near R0

Example 3: Why FRET is so distance-sensitive

Distance doubled from R₀ to 2R₀

Result: Efficiency falls from 50% to 1.5%

The sixth-power dependence means a twofold distance change alters efficiency 64-fold, which is what makes FRET a precise ruler in its narrow window.

Example 4: Acceptor photobleaching

Bleach acceptor, donor signal rises

Result: Internal control

Donor recovery is proportional to the FRET previously occurring, measured in the same cell without needing a separate donor-only sample.

Common Mistakes

⚠️

Interpreting FRET outside the 0.5–1.5 R₀ window

Efficiency saturates near 100% below and approaches zero above. Distances outside that range cannot be resolved.

⚠️

Assuming κ² = 2/3 for rigidly attached fluorophores

That value assumes free rotation. Constrained dipoles can give values from 0 to 4, introducing substantial distance error.

⚠️

Using donor quenching without a proper control

It requires a matched donor-only sample. Acceptor photobleaching provides an internal control within the same specimen.

⚠️

Ignoring spectral bleedthrough

Donor emission detected in the acceptor channel inflates apparent FRET. Correction using single-labelled controls is essential.

Frequently Asked Questions

FRET sensitivity range?

FRET is useful for distances 0.5-2× R₀ (typically 2-12 nm). At r<0.5R₀: E≈100% (insensitive). At r>2R₀: E≈0% (no FRET). E=R₀⁶/(R₀⁶+r⁶). This makes FRET a 'molecular ruler' for protein conformational changes, protein-protein interactions at nanometer scale.

Common FRET pairs?

CFP/YFP: classic genetically encoded pair, R₀=4.9nm. GFP/mCherry: R₀=5.4nm. Cy3/Cy5: organic dyes, R₀=5-6nm, used in single molecule FRET. AlexaFluor pairs: bright, photostable. For multiplexing: cascade FRET with three dyes.

Why is FRET so distance-sensitive?

Efficiency depends on the inverse sixth power of separation, so doubling the distance reduces efficiency 64-fold.

What distance range does FRET measure?

Roughly 0.5 to 1.5 times R₀, typically 2 to 10 nm. Outside that window efficiency saturates or approaches zero.

What is the orientation factor?

κ² describes relative dipole orientation. It is assumed to be 2/3 for freely rotating fluorophores but can range from 0 to 4 when they are constrained.

Why use acceptor photobleaching?

It provides an internal control within the same specimen. Donor fluorescence recovers in proportion to the FRET that was occurring.